Use of silk number assay to screen for genes enhancing corn yield

ABSTRACT

The invention provides a rapid and efficient method and assay for monitoring yield enhancement in a plant using a measure of silk number. The plant is transformed with a prospective gene associated with yield enhancement. The transformed plant is grown along with non-transformed control plants until silk growth is apparent. The change in the number of silks in the transformed plant correlated to the change in yield for the plant. Therefore, changes in yield enhancement can be estimated in the transformed plant prior to harvest of mature plant tissues.

CROSS REFERENCE

This utility application is a continuation application of U.S. Ser. No.13/602,548, filed Sep. 4, 2012, now U.S. Pat. No. 9,206,437, which is acontinuation-in-part of U.S. patent application Ser. No. 12/647,572,filed Dec. 28, 2009, now abandoned and claims the benefit U.S.Provisional Application Ser. No. 61/144,115, filed Dec. 29, 2008, eachof which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to plant molecular biology. Morespecifically, it relates to methods for measuring yield/yield potentialin plants.

BACKGROUND OF THE INVENTION

Seed yield is a very complex trait and can be further dissected intoseveral component traits, which are controlled by many interactingfactors and pathways. Genes contributing to a complex crop trait such asyield can be numerous, making it extremely difficult to find genes thatcould be used to enhance yield through low throughput transgenediscovery and validation process.

Additionally, grain yield in Zea mays is dependent upon the number ofovaries which are initiated, are fertilized and develop to maturity.Reduced grain production may result from, inter alia, a decrease in thenumber of kernel initials, restricted or untimely silk exsertion and/orkernel abortion during grain development. Maize silks comprise thestigmatic tissues of the flower, intercepting air-borne pollen andsupporting pollen tube growth to result in fertilization. Importantly,the process of fertilization determines kernel number and thus sets anirreversible upper limit on grain yield.

One of the important keys to successful high throughput (HTP) evaluationof yield enhancing genes is the ability to eliminate genes that do notpositively affect yield. However, measuring yield has been provendifficult because of the huge variation in yield associated withpollination and magnified plant to plant variation in greenhousestudies. This invention describes a new approach that is able tocircumvent these problems and that makes the HTP evaluation of yieldenhancing genes feasible.

SUMMARY OF THE INVENTION

Generally, it is an object of the present invention to provide methodsfor measuring yield enhancing gene expression in a growing plant. Thesurrogate T1 yield assay detailed in this invention is sensitive enoughto differentiate 10% difference in silk number between T1 transgenic andnull plants at P<0.1 in the greenhouse environment and can be used toidentify yield enhancement lead through Pioneer's high throughputtransgene evaluation system, which make it easier to screen thousands ofgenes to find a few that by themselves or in combination couldsignificantly increase yield in various environmental conditions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a HTP screening and validation methodfor identifying constructs that may be used to enhance yield or yieldpotential. The method combines the high throughput transgene functionanalysis (US Patent Application Publication Number 2007/0186313 A1) withthe high throughput T1 phenotyping described in this invention in adefined growth condition. The method uses a high throughput silk numberdetermination assay at a defined developmental stage of the test plantsto evaluate the yield potential in a defined growing condition.

The method determines the effect of transgene of yield or yieldpotential early in the transgene evaluation process. It allows us topredict the enhancement of a transgene on harvestable yield in T1generation, without going through the time consuming yield trialprocess.

T1 Plants to be Evaluated

The constructs evaluated in T1 silk number assay are selected based ontheir performance in yield related traits at T0 generation. T1 seeds aregenerated by crossing T0 hemizygous plants by fast growing, shortstature inbred.

Growing Conditions

Each T1 plant is grown in a classic 200 size pot (volume equivalent to1.7 L) labeled with a bar coded label with information about the plant'sgenetic identity, planting date and greenhouse location. The plantingdensity is 8.5″ between plants (˜72K plants/acre). T1 seeds are sown in50% Turface and 50% SB300 soil mixture at a uniform depth of 2″ from thesurface. Transgenic plants and their non-transgenic segregants wereidentified through strip test, assaying the presence of a marker genelinked with gene of interest.

Experimental Design

A nested block design with stationary blocks was used to minimizespatial variation. Experiment is blocked by events and constructs.Multiple events were evaluated for each constructs. For each event 15transgene positive plants and 11 to 12 transgene negative plants wereused. Positives and negatives are completely randomized within eachevent block. The transgene negative plants from events of the sameconstruct were pooled together and used as construct null.

Silk Cutting

Silk was harvested at 8 days after silking using a cutting device. Thesilk pieces are deposited in a 20 ml scintillation vial temporarilyattached to the cutting device. Residual silk pieces are rinsed into thevial with ˜10 ml 90% ethanol applied by hand from a squirt bottle. Thealcohol rinse here not only ensures that all silks are deposited in thevial but also reduces build up of sugary residues on the blades. Vialsare labeled by writing EU-ID on the vial or alternatively the vials canbe pre-labeled with bar codes containing info needed for sampledrecognition.

Silk Number Determination

Silk number determination was carried out using ImagePro silk countingsoftware. For each sample, the contents of the vial are poured into aglass Petri dish 7.5 cm diameter and with walls 14 mm high, placed ondark navy blue or black background under a digital CDD (Q-imaging)camera connected to a PC equipped with ImagePro. The layer of liquidshould be about 0.5 cm deep. Any contaminations such as anthers or husktips are removed. The sample is allowed to settle for 5 seconds beforethe imaging.

Other Parameters Collected During T1 Yield Assay

In addition to silk numbers, scientists collected other data forparameters such as specific growth rate and maximum total area. Thespecific growth rate is the plant growth rate during exponential growthperiod. Maximum total area are the maximum biomass based on the threedimensional imaging of Lemna Tec. Shedding and silking data are alsocollected.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagram illustrating the process from spikelet to kernel,showing the relationship between spikelet number, exposed silk numberand kernel numbers.

FIG. 2 shows graphs of the correlation between spikelet number and silknumber collected at different intervals (days after silking). FIG. 2aindicates 4 days, FIG. 2b illustrates 6 days and FIG. 2c shows 8 daysafter silking.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and is not intended to limit the scopeof the invention.

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Units, prefixes and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges recitedwithin the specification are inclusive of the numbers defining the rangeand include each integer within the defined range. Amino acids may bereferred to herein by either their commonly known three-letter symbolsor by the one-letter symbols recommended by the IUPAC-IUBMB NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. Unless otherwise provided for, software,electrical and electronics terms as used herein are as defined in TheNew IEEE Standard Dictionary of Electrical and Electronics Terms (5^(th)edition, 1993). The terms defined below are more fully defined byreference to the specification as a whole. Section headings providedthroughout the specification are not limitations to the various objectsand embodiments of the present invention.

In accordance with the present invention, methods and assays aredescribed for measuring enhanced yield by plants and for theidentification of candidate polynucleotides that modulate yield. Withoutwishing to be bound by this theory, plants having polynucleotides thatincrease silk number are believed to have increased yield and/orbiomass. Methods and assays of the present invention use a measurementof silk number as a correlated measure of yield enhancement.

The term “plant” includes but is not limited to a plant cell, a plantprotoplast, plant cell tissue culture from which a plant can beregenerated, plant calli, a plant clump and a plant cell that is intactin plants or parts of plants such as an embryo, seed, root, root tip andthe like.

A “subject plant” refers to a plant that is being screened for yieldenhancement.

A “control plant” may comprise, for example: (a) a wild-type plant,i.e., of the same genotype as the starting material for the geneticalteration which resulted in the subject plant or cell; (b) a plant ofthe same genotype as the starting material but which has beentransformed with a null construct (i.e., with a construct which has noknown effect on the trait of interest, such as a construct comprising amarker gene) or (c) a plant which is a non-transformed segregant amongprogeny of a subject plant. Thus, a “control plant” provides a referencepoint for measuring changes in phenotype of the subject plant, forexample, a difference in the number of silks formed in the control plantas compared to the number of silks formed the subject plant.

As used herein, “yield” may include reference to bushels per acre of agrain crop at harvest, as adjusted for grain moisture (15% typically formaize, for example), and the volume of biomass generated (for foragecrops such as alfalfa, and plant root size for multiple crops). Grainmoisture is measured in the grain at harvest. The adjusted test weightof grain is determined to be the weight in pounds per bushel, adjustedfor grain moisture level at harvest. Biomass may be measured as theweight of harvestable plant material generated. One skilled in the artwill be able to determine yield or biomass for a particular plant.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide or analogs thereof that havethe essential nature of a natural ribonucleotide in that they hybridize,under stringent hybridization conditions, to substantially the samenucleotide sequence as naturally occurring nucleotides and/or allowtranslation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Thus, DNAs or RNAswith backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term polynucleotide asit is employed herein embraces such chemically, enzymatically ormetabolically modified forms of polynucleotides, as well as the chemicalforms of DNA and RNA characteristic of viruses and cells, includinginter alia, simple and complex cells.

As used herein, the term “modulate”, “modulates” or “modulating” refersto a change, i.e., an increase or decrease in the silk numbercorrelating with plant yield.

Any plant may be screened using the methods and assays described herein,including but not limited to transgenics, inbreds, hybrids andnon-transformed plants. This also includes plants that have been treatedwith a mutagen, such as ethyl methanesulfonate (EMS) and the like. Inone aspect, the plant to be screened includes a candidate polynucleotidesuspected of modifying yield of the plant. In another aspect, the plantcomprises individual or combinations of genes to be screened for theireffect on yield.

Using methods or assays of the present invention, one skilled in the artwould be able to screen thousands of different plants, for example, forgene expression causing enhanced yield. The methods of the presentinvention are useful for a variety of applications. These plants may beadditionally screened for their ability to increase the yield or biomassof a plant as compared to a control. The present invention provides fora high throughput assay for screening a plurality of plants to identifya plant with enhanced yield.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise interveningsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening sequences (e.g., as in cDNA). Theinformation by which a protein is encoded is specified by the use ofcodons. Typically, the amino acid sequence is encoded by the nucleicacid using the “universal” genetic code. However, variants of theuniversal code, such as are present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolum or the ciliateMacronucleus, may be used when the nucleic acid is expressed therein.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host organism.For example, although nucleic acid sequences of the present inventionmay be expressed in both monocotyledonous and dicotyledonous plantspecies, sequences can be modified to account for the specific codonpreferences and GC content preferences of monocotyledons or dicotyledonsas these preferences have been shown to differ (Murray, et al., (1989)Nucl. Acids Res. 17:477-498). Thus, the maize-preferred codon for aparticular amino acid may be derived from known gene sequences frommaize. Maize codon usage for 28 genes from maize plants is listed inTable 4 of Murray, et al., supra.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of a native (non-synthetic), endogenous, biologically (e.g.,structurally or catalytically) active form of the specified protein.Methods to determine whether a sequence is full-length are well known inthe art, including such exemplary techniques as northern or westernblots, primer extension, S1 protection, and ribonuclease protection.See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Comparison to known full-lengthhomologous (orthologous and/or paralogous) sequences can also be used toidentify full-length sequences of the present invention. Additionally,consensus sequences typically present at the 5′ and 3′ untranslatedregions of mRNA aid in the identification of a polynucleotide asfull-length. For example, the consensus sequence ANNNNAUGG, where theunderlined codon represents the N-terminal methionine, aids indetermining whether the polynucleotide has a complete 5′ end. Consensussequences at the 3′ end, such as polyadenylation sequences, aid indetermining whether the polynucleotide has a complete 3′ end.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by human intervention. For example, apromoter operably linked to a heterologous structural gene is from aspecies different from that from which the structural gene was derived,or, if from the same species, one or both are substantially modifiedfrom their original form. A heterologous protein may originate from aforeign species or, if from the same species, is substantially modifiedfrom its original form by human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “introduced” includes reference to the incorporation of anucleic acid into a eukaryotic or prokaryotic cell where the nucleicacid may be incorporated into the genome of the cell (e.g., chromosome,plasmid, plastid or mitochondrial DNA), converted into an autonomousreplicon, or transiently expressed (e.g., transfected mRNA). The termincludes such nucleic acid introduction means as “transfection”,“transformation” and “transduction”.

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is substantially free from components that normallyaccompany or interact with it in its naturally-occurring environment.The isolated material optionally comprises material not found with thematerial in its natural environment or if the material is in its naturalenvironment, the material has been synthetically (non-naturally) alteredby human intervention to a composition and/or placed at a location inthe cell (e.g., genome or subcellular organelle) not native to theisolated material. The alteration to yield the synthetic material can beperformed on the material within or removed from its natural state. Forexample, a naturally-occurring nucleic acid becomes an isolated nucleicacid if it is altered, or if it is transcribed from DNA which has beenaltered, by means of human intervention performed within the cell fromwhich it originates. See, e.g., Compounds and Methods for Site DirectedMutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In VivoHomologous Sequence Targeting in Eukaryotic Cells; Zarling, et al.,PCT/US93/03868. Likewise, a naturally-occurring nucleic acid (e.g., apromoter) becomes isolated if it is introduced bynon-naturally-occurring means to a locus of the genome not native tothat nucleic acid.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer or chimeras thereof, ineither single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to that of naturally-occurring nucleotides (e.g., peptidenucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism or of a tissueor cell type from that organism. Construction of exemplary nucleic acidlibraries, such as genomic and cDNA libraries, is taught in standardmolecular biology references such as Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol. 152, AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al., MolecularCloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989) and CurrentProtocols in Molecular Biology, Ausubel et al., Eds., Current Protocols,a joint venture between Greene Publishing Associates, Inc. and JohnWiley & Sons, Inc. (1994).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, cells isolated from seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores. The class of plantswhich can be used in the methods of the invention include bothmonocotyledonous and dicotyledonous plants. A particularly preferredplant is Zea mays.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide or chimeras or analogsthereof that have the essential nature of a natural deoxy- orribo-nucleotide in that they hybridize, under stringent hybridizationconditions, to substantially the same nucleotide sequence as donaturally-occurring nucleotides and/or allow translation into the sameamino acid(s) as do the naturally-occurring nucleotide(s). Apolynucleotide can be full-length or a subsequence of a native orheterologous structural or regulatory gene. Unless otherwise indicated,the term includes reference to the specified sequence as well as to thecomplementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to naturally-occurring amino acid polymers, as well as toamino acid polymers in which one or more amino acid residue is anartificial chemical analogue of a corresponding naturally-occurringamino acid. The essential nature of such analogues ofnaturally-occurring amino acids is that, when incorporated into aprotein, that protein is specifically reactive to antibodies elicited tothe same protein but consisting entirely of naturally-occurring aminoacids. The terms “polypeptide”, “peptide” and “protein” are alsoinclusive of modifications including, but not limited to, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation. Further, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of proteins of the invention.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses and bacteria which comprise genes expressed inplant cells such Agrobacterium or Rhizobium. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots or seeds. Suchpromoters are referred to as “tissue preferred”. Promoters whichinitiate transcription only in certain tissue are referred to as “tissuespecific”. A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “repressible” promoter is apromoter which is under environmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue specific, tissuepreferred, cell type specific and inducible promoters constitute theclass of “non-constitutive” promoters. A “constitutive” promoter is apromoter which is active under most conditions.

As used herein “recombinant” includes reference to a cell or vector thathas been modified by the introduction of a heterologous nucleic acid orto a cell derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found in identical formwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under-expressed or notexpressed at all, as a result of human intervention. The term“recombinant” as used herein does not encompass the alteration of thecell or vector by naturally-occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without human intervention.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

EXAMPLES Example 1: Silk Counting Protocol

The method determines the effect of transgene of yield or yieldpotential early in the transgene evaluation process. It allowsprediction of the enhancement of a transgene on harvestable yield in T1generation, without going through the time consuming yield trial.

T1 Plants to be Evaluated

The constructs evaluated in T1 silk number assay are selected based ontheir performance in yield related traits at T0 generation. T1 seeds aregenerated by crossing T0 hemizygous plants by fast growing, shortstature inbred.

Growing Conditions

Each T1 plant is grown in a classic 200 size pot (volume equivalent to1.7 L) labeled with a bar coded label with information about the plant'sgenetic identity, planting date and greenhouse location. The plantingdensity is 8.5″ between plants (˜72K plants/acre). T1 seeds are sown in50% Turface and 50% SB300 soil mixture at a uniform depth of 2″ from thesurface. Transgenic plants and their non-transgenic segregants wereidentified through strip test, assaying the presence of a marker genelinked with gene of interest.

Experimental Design

A nested block design with stationary blocks was used to minimizespatial variation. Experiment is blocked by events and constructs.Multiple events were evaluated for each constructs. For each event 15transgene positive plants and 11 to 12 transgene negative plants wereused. Positives and negatives are completely randomized within eachevent block. The transgene negative plants from events of the sameconstruct were pooled together and used as construct null.

Silk Cutting

Silk was harvested at 8 days after silking using a cutting device. Thesilk pieces were deposited in a 20 ml scintillation vial temporarilyattached to the cutting device. Residual silk pieces are rinsed into thevial with ˜10 ml 90% ethanol applied by hand from a squirt bottle. Thealcohol rinse here not only ensures that all silks are deposited in thevial but also reduces build up of sugary residues on the blades. Vialsare labeled by writing EU-ID on the vial or alternatively the vials canbe pre-labeled with bar codes containing info needed for sampledrecognition.

Silk Number Determination Silk number determination was carried outusing ImagePro silk counting software. For each sample, the contents ofthe vial are poured into a glass Petri dish 7.5 cm diameter and withwalls 14 mm high, placed on dark navy blue or black background under adigital CDD (Q-imaging) camera connected to a PC equipped with ImagePro.The layer of liquid should be about 0.5 cm deep. Any contaminations suchas anthers or husk tips are removed. The sample is allowed to settle for5 seconds before the imaging.Other Parameters Collected During T1 Yield Assay

In addition to silk numbers, scientists collected other data forparameters such as specific growth rate and maximum total area. Thespecific growth rate is the plant growth rate during exponential growthperiod. Maximum total area are the maximum biomass based on the threedimensional imaging of Lemna Tec. Shedding and silking data are alsocollected.

Example 2: Assessing the Correlation Between Silk Number and SpikeletNumber Using a Dwarf Non-Transgenic Maize Line

As shown in FIG. 1, silk number represents potential kernel number of aplant and can be used to represent the realizable yield potential underdefined environmental condition. Kernel set is the result of a reductionfrom this potential as pollination and kernel abortion under the sameenvironmental condition impact the success of an individual plant toproduce kernels.

Assessing the Repeatability and Reproducibility of the Silk CountingSoftware

ImagePro Plus software was used to count the silk number. To assess therepeatability and reproducibility of the silk counting equipment, thesilk number was counted multiple times and the instrumental variabilitywas calculated. The variability of silk number was found to be less than0.5% using the same sample multiple times by a single operator ormultiple operators. The silk number determined by silk counting softwarecorrelated extremely well (R²=0.99) with silk number determined by handcounting.

Silking Cutting Time

Several experiments were carried out to see whether the silk number isconsistent enough among plants to be used effectively in an assayexperiment. Results from these experiments showed a significantcorrelation between silk number and spikelet number, another goodindicator of yield potential, further confirming the validity of silknumber as an indicator of yield potential. Silk counting experimentswere carried out for FAST corn maize plants under three differentfertilizer application schemes and at 3 different dates (4, 6 and 8 daysafter silking). The experiment results showed that the correlationbetween silk number and spikelet number was best when fertilizer wereapplied once every two days or when silk counting was carried out at 8days after silking (FIG. 2). As shown in Table 1, the variation of silknumber among plants was lowest when silk counting was carried out 8 daysafter silking.

TABLE 1 Silk number and their CV at different silk cutting dates Silkcutting time Silk Standard Coefficient of (days after silking) numberdeviation Variation 4 217 60.07 27.68 6 229 73.97 32.3 8 246 60.93 24.77

Example 3: T1 Silk Number Assay on Transgenic Lines to IdentifyConstructs that would Enhance Yield/Yield Potential

To assess the efficacy of the T1 yield assay, 15 potential yieldenhancement constructs selected based on their T0 performance in yieldrelated traits were evaluated. Three events were evaluated for eachconstruct. For each event, 15 transgene positive plants and 11-12transgene negative plants were used. A nested block design withstationary blocks was used to minimize spatial variation. Experimentswere blocked by events and constructs. Positives and negatives werecompletely randomized within each event block. Transgene negative plantsfrom all events of a single construct then were pooled together and usedas construct null.

As summarized in Table 2, among the fifteen GOI (gene of interest)constructs assayed, six constructs were able to significantly increasethe yield potential as defined by silk number. Two of the constructssignificantly decreased the yield potential. The assay also showed thatthe increased yield potential was not always associated with plantgrowth or biomass. Two of the constructs assayed (PHP27561 and PHP30542)have very close correlation with either specific growth rate or maximumarea or both.

TABLE 2 Percent change in silk number, specific growth rate and maximumarea Silk number Maximum area GOI % Specific growth rate % construct IDchange P value % change P value change P value PHP22743 11.2 NS 7.20.0191 2.59 NS PHP24878 −11.3 −0.0575 −0.1 NS 3.6 NS PHP25433 −6.73−0.0447 3.6 0.0269 −3.89 −0.0158 PHP25445 10.83 0.0079 −1.5 NS 0.5 NSPHP25872 4.1 NS 0.7 NS 5.3 NS PHP27215 −2.97 NS 1.4 NS 2.14 NS PHP275409.07 0.0646 1 NS −0.44 NS PHP27561 20.16 0.0020 10.80 0.0001 5.44 0.0447PHP27893 0 NS 0.55 NS 1.02 NS PHP27958 12.5 0.0339 −1.2 NS −2.4 NSPHP28098 4.29 NS −0.6 NS 3.26 0.0708 PHP28100 23.29 0.0000 0.6 NS 4.630.0257 PHP28126 2.42 NS 1.6 NS −0.57 NS PHP30542 15.55 0.0427 4.9 0.097711.5 0.0417 PHP32787 −3.6 NS 0.4 NS −2.53 −0.0407

Table 3 shows the data for the three individual PHP27561 events assayed.Among the three events assayed, two of the events have significantlyincreased silk numbers. Importantly, the increased silk number is highlycorrelated with the expression of GOI in the construct.

TABLE 3 T1 silk number assay summary for PHP27561 Specific growth GOIRelative Silk number rate Maximum area construct GOI % % % P ID eventexpression change P value change P value change value PHP27561 all 20.160.0020 20.16 0.0001 5.54 0.0447 1 −0.13 4.03 NS 3.79 NS 4.84 NS 2 5.0024.51 0.0115 6.85 0.0085 3.19 NS 3 6.28 25.75 0.0347 18.58 0.0009 30.020.0093

Example 4: The Comparison of T1 Yield Assay Results with Yield TrialResults in the Field

To assess the efficacy of T1 yield assay, 8 constructs wereretransformed into elite maize. Single copy transgenic events withtransgene expression were selected for fixing transgene and for seedincrease. Top cross seeds were generated for yield trial. All events andcontrols were randomized in an Alpha array across the replicates. 10% ofisogenic controls (construct null) are included in the trial. The yieldof these transgenic events was analyzed against their construct nulls.

As summarized in Table 4, among the 8 constructs evaluated in both T1yield assay and yield trials, six constructs showed very goodcorrelation between T1 assay results and yield trial results. All fourconstructs that did not perform well in GH did not perform well in yieldtrial. Half of the constructs that performed positively or somewhatpositively in T1 yield assay have good performances in yield trial. TheT1 yield assay seems to be a very effective lead discovery andvalidation tool.

TABLE 4 Comparison of T1 yield assay and yield trial results in thefield (Significant when greater or less than control at p < 0.05)Constructs T1 yield assay Yield trial in the field PHP25433Significantly less silks and Significantly less yield shorter ears atsilk suiting compared with control Null PHP25872 No effect on silknumber and No effect on yield ear length PHP27495 No effect on silknumber and No effect on yield ear length PHP27540 No effect on silknumber and No effect on yield ear length PHP27561 Significantly moresilks and No effect on yield longer silk cutting PHP28100 Significantlymore silks and Significantly more yield longer ears at silk cuttingcompared with control Null PHP30532 Significantly more silks andSignificantly more yield longer ears at silk cutting compared withcontrol Null PHP30542 Significantly more silks and No effect on yieldlonger ears at silk cutting

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, patent applications andcomputer programs cited herein are hereby incorporated by reference.

What is claimed is:
 1. A method for screening for potential enhancedyield in maize, comprising: a. growing a maize plant under plant growingconditions until silks develop, wherein the maize plant comprises aheterologous polynucleotide encoding a polypeptide; b. counting thenumber of silks present in the maize ear; c. comparing the number ofsilks in the maize plant with the heterologous polynucleotide with acontrol maize plant that does not comprise the heterologouspolynucleotide; and d. identifying the maize plant with an increasedsilk number when compared to the control maize plant, wherein saididentified maize plant has potentially enhanced yield.
 2. The method ofclaim 1, wherein the maize plant is an inbred or a hybrid.
 3. The methodof claim 1, wherein a plurality of maize plants are screened.
 4. Themethod of claim 3, wherein the maize plants are grown in a greenhouse.5. The method of claim 1, wherein the maize plant is a dwarf maizeplant.